DESCRIPTION: The overall aim of this project is to investigate the roles of nitric oxide (NO), cGMP, and cGMP dependent protein kinase (PKG) in long-term potentiation (LTP) at the Schaffer collateral synapse in the hippocampus. While there is general agreement that LTP at this synapse is initiated by a rise in postsynaptic Ca entering through either NMDA receptors or, under some conditions, voltage-gated Ca channels, the mechanisms for expression of the LTP are highly controversial. There is evidence to suggest that expression involves changes in postsynaptic glutamate receptors, while other data suggest that LTP involves increases in presynaptic transmitter release. Expression mechanisms therefore are argued to be either postsynaptic or presynaptic with few people arguing both. The PI and his collaborators in this center are clearly in the presynaptic camp. The implication for a presynaptic expression mechanism is that there must be some form of communication between the postsynaptic sensing of a rise in Ca and the presynaptic change in transmitter release. A number of so-called retrograde messengers have been implicated in this process. These include NO, CO, AA, platelet activating factor, and neurotrophins. The PI and collaborators have amassed a considerable body of evidence to suggest that NO plays a role as a retrograde messenger, and this grant is intended to further explore this role and to identify the downstream targets of NO such as cGMP and PKG in mediating the presynaptic changes in transmitter release. This group has also identified various phases to the expression of LTP. The late phase (L-LTP), which begins about 90 min after induction and requires 3 or 4 trains of 100 Hz, 1 sec stimulation, is protein and RNA synthesis dependent while the early phase (E-LTP), which occurs during approximately the first 60 min after induction and is induced with 1 train of stimulation, is not. There is also a recently described intermediate phase, which is dependent on PKA but not protein synthesis, although this project does not specifically address this intermediate phase. The two specific aims for this project are to explore further the role of NO, cGMP, and PKG in early and late phase LTP at the Schaffer collateral synapse in hippocampus. For early LTP, the PI and his colleagues propose: 1) to examine LTP in mice with targeted deletions or overexpression of NO synthase or soluble guanylyl cyclase; 2) to test the site of action of these molecules during LTP using intracellular injection methods in hippocampal slices; 3) to examine the cellular localization of these molecules using immunocytochemistry; 4) to examine the roles of these molecules in the induction and expression of LTP using electrophysiological and imaging techniques in cultured hippocampal neurons where the pre- and postsynaptic neurons are accessible for study. For late LTP they propose: 1) to investigate whether NO has different sites of action for late vs. early LTP; 2) to determine some of the downstream targets of NO for late LTP, focusing on two candidate for which they have preliminary evidence, ryanodine receptors and CREB.